Contact Allergens Formed on Air Exposure of Linalool. Identification

Chem. Res. Toxicol. 2004, 17, 1697-1705

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Contact Allergens Formed on Air Exposure of Linalool. Identification and Quantification of Primary and Secondary Oxidation Products and the Effect on Skin Sensitization Maria Sko¨ld,†,‡ Anna Bo¨rje,† Elma Harambasic,† and Ann-Therese Karlberg*,† Department of Chemistry, Dermatochemistry and Skin Allergy, Go¨ teborg University, SE-412 96 Go¨ teborg, Sweden, and National Institute for Working Life, SE-113 91 Stockholm, Sweden Received June 29, 2004

Linalool (3,7-dimethyl-1,6-octadien-3-ol) is an important fragrance chemical, frequently used in scented products because of its fresh, flowery odor. Linalool is an unsaturated hydrocarbon and is therefore susceptible to oxidation in the presence of air. The primary oxidation products, that is, hydroperoxides, formed in the autoxidation process, are reactive compounds that can be suspected to act as sensitizers. In the present investigation, we studied the autoxidation of linalool with emphasis on the formation of hydroperoxides. The oxidation products were isolated using flash chromatography and preparative HPLC and were identified with NMR and GC/ MS, using synthesized reference compounds. Two hydroperoxides and several different secondary oxidation products were identified, among which some contain structural features that make them potential allergens. The amounts of linalool and the major oxidation products were quantified over time, using GC and an HPLC-method, suitable for the analysis of thermolabile primary oxidation products. The hydroperoxide 7-hydroperoxy-3,7-dimethylocta1,5-diene-3-ol was found to be present in 15% in an oxidized sample. The local lymph node assay (LLNA) was used to investigate the sensitizing potential of pure linalool, two samples of air-exposed linalool, and oxidation products of linalool (an R,β-unsaturated aldehyde, a mixture of two hydroperoxides, and an alcohol). Pure linalool showed no sensitizing potential. The air-exposed samples of linalool produced clearly positive responses, and the hydroperoxides were the strongest allergens of the tested oxidation products. The study demonstrates the importance of autoxidation on the sensitizing potential of linalool. We also conclude that the sensitizing potential differs with the composition of the oxidation mixture and thus with the air exposure time.

Introduction Linalool (3,7-dimethyl-1,6-octadien-3-ol) 1 (Figure 1) occurs naturally in large amounts in various plant materials, for example, in lavender (1). It is an important fragrance chemical, widely used in scented products because of its fresh, flowery odor. Linalool was detected in 97% of deodorants on the European market (2) and in 61% of domestic and occupational products (3). Linalool was also found to be the most frequently incorporated fragrance chemical when large numbers of fragranced products (perfumes, cosmetics, household products, and soaps) were analyzed in the Netherlands and in the U.S. (4, 5). In both studies, linalool was found to be present in about 90% of the analyzed products. It is well known that terpenes can autoxidize when exposed to air. We have previously investigated the allergenic activity of pure and air-exposed linalool in guinea pigs. We found that pure linalool was not a contact allergen while air-exposed linalool sensitized the animals (6). We have also demonstrated that autoxidized linalool * To whom correspondence should [email protected]. † Go ¨ teborg University. ‡ National Institute for Working Life.

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is a frequent cause of contact allergy in consecutive dermatitis patients (7). A contact allergen is normally an electrophilic compound that can penetrate the skin barrier and react with macromolecules (proteins) in the skin to form the full antigen. Linalool is not electrophilic and should consequently not possess any contact allergenic activity. However, compounds formed in the autoxidation process incorporate oxygen in their structures, and electrophilic or radical reactive sites are created which make the oxidation products potential allergens. Because a large population is exposed to linalool, it is important to investigate the allergenic activity of the autoxidation products. Only a few studies on the autoxidation of linalool have been published (6, 8-12). Most of these studies were conducted at elevated temperatures for short periods of time, and the analyses of the oxidized materials were performed at high temperatures with GC using the split/ splitless technique, and identifications of oxidation products were performed by mass spectrometry (MS). At high temperatures, thermolabile autoxidation products are decomposed. Therefore, hydroperoxides of linalool were not described before we reported the presence of linalool hydroperoxide 4 in a sample of air-exposed linalool (6).

10.1021/tx049831z CCC: $27.50 © 2004 American Chemical Society Published on Web 11/25/2004

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Sko¨ ld et al.

Figure 1. Structures of linalool, synthesized reference compounds 3-11, and identified oxidation products in air-exposed linalool 2-9.

In that study, we used a GC method with on-column injection at 35 °C and a temperature program with a maximum temperature of 185 °C, but also using this method some decomposition of the hydroperoxide was seen. The aim of the present study was to investigate the autoxidation of linalool under conditions similar to the room-temperature storage of fragrance raw materials and fragranced products. Because we wanted to be able to isolate, identify, and quantify the thermolabile primary oxidation products, that is, hydroperoxides, an HPLC method, suitable for the analysis of such compounds, was developed. We also wanted to determine the sensitizing potential of pure and oxidized linalool and of some oxidation products of linalool with different functional groups using the local lymph node assay (LLNA).

Experimental Procedures Caution: Oxidized linalool contains contact allergenic substances, and therefore skin contact must be avoided and the compounds must be handled with care. Chemicals. Linalool (3,7-dimethyl-1,6-octadien-3-ol) 1, 97% was purchased from Lancaster Synthesis (England) or SigmaAldrich Chemie (Germany) and was used without further purification in the air exposure experiments. Acetone was purchased from Merck (Germany), and olive oil was purchased from Apoteket AB (Sweden). Instrumentation and Mode of Analysis. Chromatographic separations were performed using Merck or Scharlau silica gel 60 (230-400 mesh ASTM). TLC plates (Merck, 60 F254 silica gel) were, after development, sprayed with a visualizing reagent containing anisaldehyde, sulfuric acid, and acetic acid in ethanol. GC analyses were performed using a Hewlett-Packard (HP) 6890 gas chromatograph equipped with an on-column injector and a flame ionization detector, using a 30 m fused silica column (HP-5; ID 0.25 mm, 0.25 µm film thickness) and nitrogen as carrier gas. The column temperature was 35 °C at injection, held isothermally for 2 min, raised to 185 °C at a rate of 5 °C/min, and finally held at 185 °C for 5 min. The detector temperature was 250 °C, and 1,2,3,5-tetramethylbenzene was used as internal standard. Electron impact (EI) mass spectral data (70 eV) were obtained on an HP mass selective detector 5973 (scanned m/z: 50-500), connected to a GC HP 6890. HPLC analyses were conducted on Merck LaChrom 2000 instrument with a diode array detector. Preparative HPLC was performed using a Gilson pump model 305 and a Gilson UV/VIS detector model 119. 1H and 13C NMR spectra were recorded on a JEOL eclipse+ 400 MHz spectrometer using CDCl3 as solvent. Chemi-

cal shifts (δ) are reported in ppm relative to CHCl3 at δ 7.25 and δ 77.0 for 1H and 13C spectra, respectively. IR spectra were obtained on a Perkin-Elmer FT-IR PC 16 spectrophotometer. Photooxidation was performed using a Rayonet reactor equipped with 16 RPR 350 nm lamps. Air Exposure Procedure. Linalool (Lancaster) was airexposed in an Erlenmeyer flask, covered with aluminum foil to prevent contamination. It was stirred for 1 h, four times a day for 80 weeks, as previously described (13). Samples were taken on a regular basis and stored in the freezer under nitrogen, to be analyzed with GC and HPLC to determine the concentrations of linalool and the major oxidation products. Oxidized samples were also flash chromatographed to isolate and identify the oxidation products formed. Isolated compounds were characterized with NMR and GC/MS. Isolation of Major Oxidation Products Using Flash Chromatography. Linalool, that had been air-exposed for different periods of time, was subjected to flash chromatography on silica gel columns. Starting from about 5 g of oxidized material, repeated purifications were made. Mixtures of nhexane and ethyl acetate were used for elution, starting with 10% ethyl acetate in hexane, whereafter the proportion of ethyl acetate was increased. The purity of isolated compounds was controlled with GC. Isolation of Minor Oxidation Products by Preparative HPLC. Oxidized linalool was fractionated by flash chromatography, whereafter the fractions were subjected to preparative HPLC. A Nucleosil 50-7 preparative column (20 × 250 mm, 7 µm particles, Macherey-Nagel) was used. The eluent consisted of 5% 2-propanol, 35% tert-butyl methyl ether, and 60% nhexane, and the flow rate was 20 mL/min. The compounds were monitored at 205 and 230 nm. HPLC Analysis. An analytical silica column, Purospher STAR (46 × 250 mm, 5 µm particles, Merck), was used. The temperature of the column and the sample rack in the auto sampler was set to 20 °C. Mobile phase: 40% tert-butyl methyl ether and 60% n-hexane isocratic elution for 10 min, a linear gradient for 5 min reaching 60% tert-butyl methyl ether and 40% n-hexane, isocratic elution with 60% tert-butyl methyl ether and 40% n-hexane for additional 15 min. The flow rate was 1 mL/min. Blank subtractions of the gradient were performed. Retention times for synthesized and isolated compounds were determined and compared (Figure 3). Syntheses of Reference Compounds. 2,2,6-Trimethyl6-vinyltetrahydro-2H-pyran-3-ol (3). The synthesis was performed as described in the literature (14) and gave 3 in 18.4% yield. 1H and 13C NMR data are available in the literature. EIMS m/z 94.1(87), 83.1(16), 79.1(24), 69.1(16), 68.1(100), 67.1(56), 59.1(66), 55.1(18) and 94.1(79), 83.1(16), 79.1(23), 69.1(15), 68.1(100), 67.1(57), 59.1(61), 55.1(19).

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Figure 2. Concentrations of linalool and the major hydroperoxide 7-hydroperoxy-3,7-dimethylocta-1,5-diene-3-ol 4 in airexposed linalool, over time. Quantification of linalool was performed with GC using the on-column technique. For the hydroperoxide, HPLC was used. 2,6-Dimethylocta-3,7-diene-2,6-diol (5) (Scheme 1). (3Methyl-but-2-enyl)-phenyl Sulfide (15). Anhydrous K2CO3 (5.0 g, 36.3 mmol) and thiophenol (1.9 mL, 18.2 mmol) were added to DMF (50 mL) at 0 °C. 3,3-Dimethyl allyl bromide (2.3 g, 15.1 mmol) was added, and the reaction mixture was stirred for 5 h. Diethyl ether was added, and the resulting solution was washed with NH4Cl (3 × 50 mL), NaHCO3 (3 × 50 mL), and brine (50 mL). Concentration and purification by flash chromatography (starting with 17% ethyl acetate/n-hexane, and increasing the proportion of ethyl acetate) and bulb-to-bulb distillation afforded 15 (1.7 g) in 64% yield. 1H NMR δ 7.357.16 (5H, m), 5.30 (1H, t, J ) 7.7 Hz), 3.54 (2H, d, J ) 7.7 Hz), 1.71 (3H, s), 1.58 (3H, s); 13C NMR δ 136.8, 136.3, 129.7, 128.7, 125.9, 119.3, 32.2, 25.6, 17.6. 3-Methyl-2-butenyl Phenyl Sulfoxide (16). The oxidation of the sulfide 15 with H2O2 and AcOH was performed as described in the literature (15). 1H NMR δ 7.61-7.48 (5H, m), 5.06 (1H, t, J ) 7.9 Hz), 3.61-3.49 (2H, m), 1.71 (3H, s), 1.40 (3H, s); 13C NMR δ 143.4, 142.4, 131.1, 128.9, 124.5, 111.1, 56.7, 26.0, 18.1. IR (KBr) 1045.28 (SdO) cm-1. 2,6-Dimethylocta-3,7-diene-2,6-diol (5). The synthesis was performed as described in the literature (16), starting from 16. 1H NMR δ 5.91 (1H, dd, J ) 17.6, 10.2 Hz), 5.73-5.58 (2H, m), 5.20 (1H, d, J ) 17.6), 5.06 (1H, d, J ) 10.6), 2.32-2.20 (2H, m), 1.31 (6H, s), 1.27 (3H, s); 13C NMR δ 144.7, 142.7, 121.7, 112.0, 72.6, 70.7, 45.0, 29.9, 29.8, 27.4. EIMS m/z 82.1(100), 71.1(81), 67.1(57), 55.1(21). 6-Hydroxy-2,6-dimethylocta-2,7-dienal (6). t-BuOOH, 5.06.0 mM in decane (11.8 mL, 64.8 mmol), and SeO2 (1.8 g, 16.2 mmol) were added to 50 mL of CH2Cl2 and stirred at room temperature. Linalool (5.0 g, 32.4 mmol) was added slowly, and the solution was stirred for 2.5 h. The organic phase was washed with 25 mL of 10% KOH, 25 mL of Na2O3S2 (aq), and 25 mL of brine. Concentration and purification by flash chromatography (20% ethyl acetate/n-hexane) afforded 6 in 12% yield. 1H NMR δ 9.35 (1H, s), 6.48 (1H, tq, J ) 7.4, 1.1 Hz), 5.89 (1H, dd, J ) 17.4, 10.8 Hz), 5.23 (1H, dd, J ) 17.2, 1.1 Hz), 5.09 (1H, dd, J ) 10.6, 1.1 Hz), 2.38 (2H, m), 1.75-1.63 (2H, m), 1.71 (3H, s), 1.31 (3H, s); 13C NMR δ 195.3, 154.7, 144.3, 139.2, 112.4, 72.9, 40.3, 28.1, 23.8, 9.1. EIMS m/z 96.1(76), 95.1(48), 93.1(26), 91.1(20), 87.1(18), 83.1(19), 82.1(23), 81.1(19), 79.1(19), 71.1(68), 70.1(36), 69.1(15), 68.1(19), 67.1(80), 65.1(25), 55.1(100), 53.1(36). 2,6-Dimethylocta-1,7-diene-3,6-diol (7). The synthesis was performed as described in the literature (17) in four steps, starting from linalyl acetate, and gave 7 in 7% yield as a mixture of diastereomers. 1H NMR data agree with published data. 13C NMR δ (147.4, 147.3), (144.9, 144.8), (112.0, 111.9), (111.0, 110.9), (76.2, 75.7), 72.9 (38.3, 37.7), (29.3, 29.1), (28.3, 28.0),

Figure 3. HPLC chromatograms of a sample of oxidized linalool (a), a mixture of synthesized reference compounds that are present in oxidized linalool (b), and two synthesized reference compounds that were not found in oxidized linalool (c). The separations were monitored at 205 nm, and blank subtractions were performed to get a straight baseline. (17.9, 17.8). EIMS m/z 137.1(16), 82.1(58), 81.1(23), 79.1(16), 71.1(79), 69.1(21), 68.1(36), 67.1(100), 55.1(34). 2,6-Dimethylocta-2,7-diene-1,6-diol (8). The synthesis was performed using the procedure described by Liu et al. (17). Linalyl acetate was oxidized to the allylic alcohol using SeO2 and tBuOOH. The acetate group was then removed with anhydrous K2CO3 in methanol and gave 8 as a colorless oil in 17% yield. 1H NMR δ 5.91 (1H, dd, J ) 17.4, 10.8 Hz), 5.41 (1H, tq, J ) 7.2, 1.3 Hz), 5.21 (1H, dd, J ) 17.2, 1.1 Hz), 5.07 (1H, dd, J ) 10.6, 1.1 Hz), 3.98 (2H, s), 2.07 (2H, m), 1.65 (3H, s), 1.61-1.54 (2H, m), 1.29 (3H, s); 13C NMR δ 144.8, 134.9, 125.8, 112.0, 73.3, 68.5, 41.6, 27.7, 22.3, 13.6. 7-Hydroperoxy-3,7-dimethylocta-1,5-diene-3-ol (4) and 6-Hydroperoxy-3,7-dimethylocta-1,7-diene-3-ol (9). Linalool (0.58 g, 3.8 mmol) was added to a CHCl3 solution of tetrabutyl-

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Chem. Res. Toxicol., Vol. 17, No. 12, 2004 Scheme 1. Synthesis of 2,6-Dimethylocta-3,7-diene-2,6-diol

ammonium salt of Bengal rose, prepared according to a procedure described by Ba¨ckstro¨m et al. (18). The solution was irradiated for 5 h using a Rayonet reactor and a constant flow of oxygen. The solvent was removed under reduced pressure. The residue was dissolved in ether and filtered through a plug of SiO2 to remove the Bengal rose. The ether was removed, and the crude product was purified by flash chromatography (20:80 etyl acetate/hexane). This resulted in a colorless oil containing a 5:3 mixture of 4 and the two diastereoisomers of 9, in total of 68% yield (0.43 g). Compound 4. 1H NMR δ 5.91 (1H, dd, J ) 17.2, 10.6), 5.68 (1H, ddd, J ) 15.8, 7.2, 6.3), 5.61 (1H, d, J ) 16.0), 5.19 (1H, dd, J ) 17.2, 1.2), 5.06 (1H, dd, J ) 10.6, 1.2), 2.31 (1H, dd, J ) 13.6, 6.3), 2.26 (1H, dd, J ) 13.6, 7.1), 1.32 (6H, s), 1.28 (3H, s); 13C NMR δ 144.7, 137.9, 126.6, 112.2, 82.0, 72.7, 45.2, 27.7, 24.4, 24.3. Compound 9. 1H NMR δ 5.85 (1H, dd, J ) 17.3, 10.8), 5.86 (1H, dd, J ) 17.3, 10.6), 5.20 (1H, dd, J ) 17.4, 1.1), 5.19 (1H, dd, J ) 17.4, 1.1), 5.05 (2H, dd, J ) 10.8, 1.1), 4.99 (4H, m), 4.28 (2H, m), 1.70 (6H, bs), 1.66-1.49 (8H, m), 1.27 (bs, 6H); 13C NMR δ (144.6, 144.5), (143.5, 143.4), (114.3, 114.2), (112.2, 112.1), (89.6, 89.4), (73.1, 73.0), (37.8, 37.7), (28.1, 28.0), (25.1, 25.0), (17.6, 17.5). 6-Hydroxy-2,6-dimethylocta-2,7-dien-4-one (10). The synthesis was performed as described in the literature (19). 1H NMR δ 6.01 (1H, m), 5.91 (1H, dd, J ) 17.4, 10.8), 5.20 (1H, dd, J ) 17.2, 1.1), 5.02 (1H, dd, J ) 10.6, 1.1), 2.67 (2H, J ) 16.5 Hz, AB), 2.13 (3H, d, J ) 1.1), 1.89 (3H, d, J ) 1.5), 1.3 (3H, s); 13C NMR δ 201.7, 157.8, 144.2, 124.4, 112.2, 73.4, 52.6, 28.4, 27.9, 21.1. 3,7-Dimethylocta-1,6-diene-3,5-diol (11). The synthesis was performed as described in the literature (19) and gave 11 in 82% yield, as a diastereomeric mixture. 1H NMR δ 5.94 (1H, dd, J ) 17.2, 10.6 Hz), 5.22 (1H, dd, J ) 17.2, 1.5 Hz), 5.235.19 (1H, m), 5.16 (1H, dd, J ) 10.6, 1.5 Hz), 4.75 (1H, ddd, J ) 9.9, 8.5, 3.3 Hz), 1.79 (1H, dd, 14.6, 9.8 Hz), 1.70 (3H, d, J ) 1.1 Hz), 1.68 (3H, d, J ) 1.5 Hz), 1.55 (1H, dd, J ) 14.6, 3.3 Hz), 1.38 (3H, s) and 5.92 (1H, dd, J ) 17.2, 10.8 Hz), 5.37 (1H, dd, J ) 17.2, 1.8 Hz), 5.23-5.19 (1H, m), 5.02 (1H, dd, J ) 10.8, 1.5 Hz), 4.61 (1H, ddd, J ) 8.8, 8.4, 2.2 Hz), 1.81 (1H, dd, J ) 14.2, 8.5 Hz), 1.68 (3H, d, J ) 1.5 Hz), 1.63 (3H, d, J ) 1.1 Hz), 1.52 (1H, dd, J ) 14.2, 2.2 Hz), 1.28 (3H, s); 13C NMR δ (145.8,

Sko¨ ld et al. 144.0), (134.8, 134.6), (127.8, 127.7), (112.6, 111.3), (74.0, 73.3), (67.3, 66.3), (47.2, 47.0), (30.1, 26.7), (25.7, 25.6), (18.2, 18.1). EIMS m/z 134.1(14), 119.1 (23), 91.1(24), 85.1(21), 83.1(24), 82.1(78), 79.1(21), 71.1(50), 70.1(20), 68.1(34), 67.1(100), 55.1(56), 53.1(19) and 134.1(17), 119.1(27), 91.1(27), 85.1(24), 83.1(24), 82.1(82), 81.1(15), 79.1(22), 77.1(15), 71.1(59), 70.1(19), 68.1(33), 67.1(100), 65.1(16), 55.1(57), 53.1(20). Quantification of Linalool and Oxidation Products. Quantification of Linalool, 2-(5-Methyl-5-vinyltetrahydrofuran-2-yl)propan-2-ol (2), and 2,2,6-Trimethyl-6-vinyltetrahydro-2H-pyran-3-ol (3). The amounts of linalool, 2, and 3 in the air-exposed samples were determined using GC. Analyses were made on pure reference compounds with added internal standard (1,2,3,5-tetramethylbenzene) to determine the response factors. The same amount of internal standard was added to the dissolved samples of oxidized linalool. Using the response factor, the amount of each compound in the samples could be determined. Quantification of 7-Hydroperoxy-3,7-dimethylocta-1,5diene-3-ol (4). The amount of hydroperoxide 4 was determined using the HPLC method with an analytical silica column, Purospher STAR (46 × 250 mm, 5 µm particle size, Merck). Mobile phase: 40% tert-butyl methyl ether and 60% n-hexane. The isolated hydroperoxide was used to make an external calibration curve from which the concentrations of the hydroperoxide in the oxidized linalool samples could be determined. Sensitization Experiments in Mice. Female CBA/Ca mice, 8 weeks of age, were purchased from Harlan (Netherlands). The animals were housed in cages with hepa-filtered airflow, under conventional conditions in light-, humidity- and temperaturecontrolled rooms. The animals were allowed to acclimate for 1 week prior to use. All animal procedures were approved by the local ethics committee. The sensitizing potential of the chemicals was investigated using the LLNA (20). Female mice (9 weeks of age) in groups of four were treated by topical application on the dorsum of both ears with 25 µL of test material or with a vehicle control. Treatments were performed daily for 3 consecutive days (days 0, 1, and 2). On day 5, following the start of treatment, all mice were injected intravenously via the tail vein with 20 µCi of [methyl-3H]thymidine (2.0 Ci/mmol, Amersham Biosciences, UK) in 250 µL of phosphate buffered saline (PBS). After 5 h, the mice were sacrificed, the draining lymph nodes were excised and pooled for each group, and single-cell suspensions of lymphnode cells in PBS were prepared using cell-strainers (Falcon, BD Labware, 70 µm pore size). Cell suspensions were washed twice with PBS, precipitated with 5% TCA, and left in the refrigerator overnight. The samples were then centrifuged, resuspended in 1 mL 5% TCA, and transferred to 10 mL of scintillation cocktail (EcoLume, INC Radiochemicals, USA). Thymidine incorporation was measured by β-scintillation counting on a Beckman LS 6000TA instrument. Results are expressed as mean dpm/lymph node for each experimental group and as stimulation index (SI), that is, test group/control group ratio. Test materials that at one or more concentrations caused an SI greater than 3 were considered to be positive in the LLNA. EC3 values (the estimated concentration required to induce an SI of 3) were calculated and used to compare the sensitizing potency of the different test materials. The EC3 values were calculated by linear interpolation (21). For treatment of the ears of the mice, the test materials were dissolved in acetone:olive oil (4:1 v/v). All solutions were prepared freshly for every application, except for the aldehyde and the alcohol solutions. These solutions were prepared on day 0 and stored in the refrigerator during the study. The hydroperoxides 4 and 9 were tested as a mixture (in an approximate ratio of 5:3) because of the impracticability of separating these two substances, in the quantities needed for the assay. To determine the sensitizing potential of pure linalool, we used a sample of linalool (Sigma-Aldrich Chemie) that was distilled the day before the start of the experiments (to eliminate any oxidation products and other impurities). The test solutions were

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Table 1. LLNAa Responses for Pure Linalool, Two Samples of Linalool Air-Exposed for 10 and 45 Weeks, Respectively, and Oxidation Products (a Mixture of Two Hydroperoxides 4 and 9, an Aldehyde 6, and an Alcohol 5) test material/concentration pure linalool (Sigma Aldrich Chemie) control 25% 50% 100% air-exposed linalool, 10 weeks control 5% 10% 25% air-exposed linalool, 45 weeks control 2.5% 10% 25% linalool hydroperoxides 4 and 9 control 0.5% 2.5% 7.5% linalool aldehyde 6 control 1% 5% 15% linalool alcohol 5 control 1% 10% 30%

DPM/lymphnode

SI

EC3 46.2

430.0 813.3 1376.3 1301.1

1.9 3.2 3.0 9.4

346.2 489.5 1110.2 4406.0

1.4 3.2 12.7 4.8

542.1 839.9 3465.7 6311.1

1.6 6.4 11.6 1.6

498.1 665.7 2123.7 3551.8

1.3 4.3 7.1 9.5

545.6 658.5 1095.5 2299.3

1.2 2.0 4.2

551.6 574.4 721.4 701.3

1.0 1.3 1.3

a Groups of mice (n ) 4) received 25 µL of the test material dissolved in vehicle (AOO 4:1) in the concentrations indicated, on the dorsum of both ears daily for 3 consecutive days. Control animals were treated in the same way with the vehicle alone. All mice were injected intravenously 5 days after the first treatment, with 250 µL of PBS containing 20 µCi of [methyl-3H]thymidine. Five hours later, draining auricular lymph nodes were excised and pooled for each group, and single-cell suspensions of lymph node cells were prepared. The thymidine incorporation was measured by β-scintillation counting. The increase in thymidine incorporation relative to vehicle-treated controls was derived for each experimental group and recorded as stimulation index. EC3 values were calculated using linear interpolation.

prepared in weight/volume %. The concentrations were chosen according to earlier experience in guinea pig tests and from literature data on substances with similar structural properties, to obtain stimulation indices that would allow calculation of EC3 values (Table 1).

Results and Discussion In the present study, we can for the first time, using the LLNA, calculate the increase of the sensitizing capacity that takes place when a pure nonoxidized compound is turned into an oxidation mixture by air exposure. We can also demonstrate that the sensitizing potential can differ depending on the composition of the oxidation mixture and thus on the time of air exposure. Although the autoxidation of terpenes is well known, our study is the first, to the best of our knowledge, that fully describes the oxidative decomposition of linalool showing the formation of both primary and secondary oxidation products in air-exposed linalool. We have identified one of the primary oxidation products, the hydroperoxide 4, as the major sensitizer constituting 15% of the oxidation

mixture that was air-exposed for 45 weeks. Hydroperoxides 4 and 9, in a mixture 5:3, gave an EC3 value of 1.6, showing them to be strong sensitizers. We have previously shown hydroperoxides to be the major sensitizers in oxidation mixtures also from other compounds with structures different from or similar to linalool, that is, hydroperoxides formed from air-exposed alcohol ethoxylates (22), from air-exposed abietic acid in colophony (23), and from air-exposed limonene (24). Air Oxidation of Linalool. Linalool was air-exposed at room temperature. Samples were taken on a regular basis for GC analysis, to determine the remaining linalool over time. The analyses showed that the amount of linalool started to decrease immediately (Figure 2). After about 30 weeks, 50% of the original compound was consumed, and after 80 weeks only about 4% remained (Figure 2). In the chromatograms, a number of peaks appeared, indicating that many compounds were formed when linalool was air oxidized. Autoxidation Products of Linalool. Air-exposed linalool was subjected to flash chromatography and preparative HPLC to isolate oxidation products. To facilitate the identification of oxidation products, several potential oxidation products were synthesized, and their chromatographic and spectral properties were compared with those of the isolated oxidation products. Six substances were isolated and identified: 2-(5-methyl-5vinyltetrahydrofuran-2-yl)propan-2-ol 2, 2,2,6-trimethyl6-vinyltetrahydro-2H-pyran-3-ol 3, 7-hydroperoxy-3,7dimethylocta-1,5-diene-3-ol 4, 2,6-dimethylocta-3,7-diene2,6-diol 5, 6-hydroxy-2,6-dimethylocta-2,7-dienal 6, and 2,6-dimethylocta-1,7-diene-3,6-diol 7 (Figure 1). The identification of the hydroperoxide 4 was performed without a reference compound, as described earlier (6). Some of the oxidation products were not possible to isolate completely pure because they were formed in low amounts and did not separate well from other oxidation products in the methods used. However, when the impure fractions were analyzed with HPLC and NMR, the retention times and spectra agreed with those of synthesized reference substances and showed the presence of 2,6-dimethylocta2,7-diene-1,6-diol 8 and 6-hydroperoxy-3,7-dimethylocta1,7-diene-3-ol 9 (Figure 1). HPLC Analysis of Autoxidized Linalool. The analytical HPLC-method gave separation of all of the identified substances and was used to determine and compare the retention times for the synthesized and the isolated compounds (Figure 3). Because monoterpenes are volatile compounds with low UV absorption, the commonly used analytical method is GC with split/splitless injection (25). Such a method only detects the more stable secondary oxidation products and not the hydroperoxides, because it is performed at high temperatures. To fully understand the oxidative decomposition of linalool, the HPLC method used here is preferred. The HPLC method is also needed to be able to quantify the content of hydroperoxides, which is important for the further investigation of the allergenic activity of oxidized linalool. Quantification of Oxidation Products in Autoxidized Linalool. The ethers in the oxidation samples were quantified using GC. The furan-derivative 2 was formed in a relatively high amount, reaching a plateau at 20% concentration after ca. 50 weeks. The pyranderivative 3 was formed to a lesser extent and reached a maximum concentration of 4% after 79 weeks. The hydroperoxide 4 in oxidized samples of linalool was

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Figure 4. Autoxidation of linalool. Abstraction of allylic hydrogen atoms in linalool gives rise to the allylic radicals 1a (trans and cis isomers) and 1c, and their resonance forms 1b and 1d. These radicals can react reversibly with triplet state oxygen to give the peroxyl radicals 2a-d. Subsequent hydrogen atom abstraction will give the hydroperoxides 4, 9, and 12-14. The hydroperoxides eventually decompose and form the corresponding secondary oxidation products.

quantified using HPLC. It was found to be formed in high amounts, and it reached a maximum concentration of about 15% after 48 weeks at which time the concentration of linalool was 26% (Figure 2). The diagram shows that, after this point, degradation is the dominating process and the concentration of the hydroperoxide decreases. Mechanism for the Formation of Autoxidation Products from Linalool. Linalool is, like other unsaturated hydrocarbons, susceptible to oxidation in the presence of air. There are three positions in the molecule where allylic hydrogen atoms can be abstracted, leading to the formation of the allylic radicals 1a (trans and cis isomers) and 1c, and their resonance forms 1b and 1d (Figure 4). These radicals can react reversibly with triplet state oxygen to give the peroxyl radicals 2a-2d. Subsequent hydrogen atom abstraction will give the hydroperoxides 4, 9, 12, 13, and 14. Electron-donating groups are known to stabilize peroxyl radicals by a hyperconjugative effect (26). The difference in alkyl substitution on the peroxyl-bearing carbons can therefore influence the

equilibrium between the alkyl radicals and their corresponding peroxyl radicals, and consequently affect the proportion of the resulting hydroperoxides in the oxidation mixture. This implies that the secondary peroxyl radical 2b would be favored over the primary peroxyl radical 2a, and the tertiary peroxyl radical 2d over the secondary peroxyl radical 2c. The hydroperoxides 4 and 9 were the only ones found in oxidized linalool, which is in accordance with them being formed from the more stable peroxyl radicals. The reason for hydroperoxide 4 being formed in higher amounts than hydroperoxide 9 is probably that the secondary hydrogen atom is more easily abstracted (leading to allylic radical 1c T 1d) than the primary hydrogen atom (leading to radical 1a T 1b). The presence of hydroperoxides 12-14 in the oxidation mixture could not be excluded. If they are formed, they are most probably rapidly converted to secondary oxidation products (22). Another possible way of conversion between hydroperoxides 9, 12, and 13, and also between hydroperoxides

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Figure 5. Proposed mechanism for the formation of the furanand pyranoxides of linalool. A secondary oxidation product that can be formed from the tertiary hydroperoxide, an epoxide, will, after attack from the hydroxyl group in linalool, give the two oxides. The ring formation is promoted by acidic conditions.

Figure 6. Dose response curves for the compounds tested in the local lymph node assay (LLNA). The concentrations are given in M. The molar concentrations of oxidized linalool are calculated using the molecular weight of linalool.

4 and 14, is rearrangement at the hydroperoxide level (Figure 4). There are several reports on the rearrangement of allylic hydroperoxides by a free-radical mechanism, in which the hydroperoxides are converted to allyl peroxyl radicals, and oxygen is transferred across the allyl groups to give the isomeric allylic hydroperoxides (27-30). The hydroperoxides that are formed in the autoxidation process are known to decompose to secondary oxidation products, for example, alcohols, ketones, aldehydes, and polymeric material. Alcohol 5 (Figure 1), which could be formed by decomposition of the main hydroperoxide 4 of linalool, was found to be the most prominent among the alcohols in the oxidation mixture. Alcohol 7 (Figure 1), corresponding to the other isolated hydroperoxide 9, was also identified in the mixture. Indirect evidence for the formation of the primary hydroperoxide 12 (Figure 4) was the detection of the alcohol 8 and the aldehyde 6 (Figure 1), which are secondary oxidation products of this hydroperoxide. The ketone 10 and the alcohol 11 (Figure 1) were synthesized as reference compounds. They would be possible degradation products from the secondary hydroperoxide 14 (Figure 4). However, neither the hydroperoxide nor the corresponding secondary oxidation products were found in the oxidation mixture. This indicates that rearrangement of the peroxyl radical 2c or the hydroperoxide 14 resulting in the most stable peroxyl radical 2d and hydroperoxide 4 is the dominating reaction pathway. A probable explanation for the formation of the pyran and furan derivatives of linalool is that the tertiary hydroperoxide 4 can form an epoxide as a secondary oxidation product (Figure 5). This epoxide is readily attacked by the hydroxyl group in linalool, on either of the two epoxide-carbons, which gives the ethers. In our attempts to synthesize the epoxide, we observed that both the pyran- and the furanoxides also were formed. Descriptions in the literature (31, 32) confirm the difficulty of forming this epoxide without obtaining the oxides and state that the ring-formation is promoted by acidic conditions (Figure 5). The pH in different states of oxidized linalool was measured, and it was found to decrease with longer oxidation times, which will favor the formation of the oxides.

Skin Sensitizing Potential. The LLNA can be used to determine the relative skin sensitizing potency of chemicals via interpolation of the quantitative dose response data generated. The sensitizing capacity of pure linalool and of the two different samples of air-exposed linalool was investigated to determine the effect of autoxidation on the allergenic activity. The results from the LLNA-experiments are presented in Table 1. The EC3 value for pure linalool was 46.2. The oxidized samples induced greater proliferation and gave EC3 values of 9.4 for linalool air-exposed for 10 weeks, and of 4.8 for linalool air-exposed for 45 weeks (Figure 6). The sensitizing capacity of some of the identified oxidation products from oxidized linalool was also investigated (Table 1). The hydroperoxides 4 and 9, and the R,β-unsaturated aldehyde 6, were suspected to be allergenic and were therefore tested in the LLNA. Alcohols are not considered to be reactive enough for proteinbinding, and they are not known to cause sensitization. Therefore, only the most prominent among the identified alcohols, alcohol 5, was chosen to be tested. We have previously shown that the pyran- and furanoxides do not possess any substantial allergenic activity (14). The hydroperoxides 4 and 9, which were tested as a mixture, were shown to be strong allergens with an EC3 value of 1.6. The potency of these hydroperoxides is in the same range as shown for other terpene hydroperoxides (14, 33). The aldehyde 6 was shown to be a weaker allergen than the hydroperoxides and gave an EC3 value of 9.5. According to the literature (34-36), aldehydes show a wide range of EC3 values (50) depending on the structure. Alcohol 5 was shown to be a nonsensitizer, as expected. Pure linalool tested in 50% concentration gave an SI value of 3.2, while the 100% concentration gave an SI value of 3.0. The response is just above the threshold for being judged as an allergen, and no clear dose response is seen. For the identification of false positive reactions in the LLNA, Basketter et al. (37) have postulated the following criteria: (1) test substance does not have a structural alert, (2) test substance is known to be a significant skin irritant, (3) dose response is odd and/or weakly positive, only at high test concentration, (4) interanimal and/or interexperiment variation is high, and

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(5) draining lymph node cells do not have surface markers characteristic of skin sensitization. In our investigations, we found a very weak response in concentrations known to cause irritation (50%, 100%) (38-40), and we did not get a clear dose-response. On the basis of this, and the fact that linalool does not have a structural alert, we consider that the effect observed for linalool is due to its irritating effect. This result is in accordance with our previous investigation of the sensitizing capacity of pure linalool in guinea pigs (6) and with reports in the literature (41). Our sensitization experiments showed that the autoxidation greatly influences the sensitizing effect of linalool. The sample of linalool air-exposed for 10 weeks gave a clearly positive result, and the sample of linalool that was air-exposed for 45 weeks showed an even stronger sensitizing capacity. The effect is illustrated in Figure 6, where the dose response curves for the different test materials are shown. The sensitizing effect of oxidized linalool is influenced by the oxidation time, and thus by the composition of oxidation products in the sample. The hydroperoxides are strong contact allergens, and their dose-response curve is steep, which means that they reach high stimulation indices over a low concentration range. The dose-response curves for the oxidized samples of linalool have the same steep slope as the doseresponse curve of the hydroperoxides, but they are shifted to the right because of lower contents of hydroperoxides. The less potent aldehyde 6 has a shallower doseresponse curve, while the nonsensitizers, alcohol 5 and pure linalool, have almost horizontal curves. In this study, we have shown the linalool hydroperoxide 4 to be a major oxidation product in oxidized linalool. The air-exposed samples used in the animal experiments were analyzed for their contents of linalool and the hydroperoxide 4. The 10 weeks air-exposed sample contained 75% linalool and 4% of the hydroperoxide, and the 45 weeks oxidized sample contained as much as 15% of the hydroperoxide (30% linalool). The concentration of hydroperoxide 9 was estimated by comparing the areas of the peaks of the two hydroperoxides in the HPLCchromatograms, and it was found to be approximately 1% in the sample air-exposed for 10 weeks and 4% in the sample air-exposed for 45 weeks. The hydroperoxides were the strongest allergens of the tested oxidation products. The sensitizing potential of the individual hydroperoxides is not known because they were tested as a mixture. The hydroperoxide 4 was present in higher concentrations and is therefore believed to be the major contributor to the increase in the sensitizing effect of linalool, during air exposure. We have, in this investigation, demonstrated the effect of autoxidation on the sensitizing potential of linalool. To study the frequency of allergic reactions to oxidized linalool, the 45 weeks oxidized sample was also used in a patch test study on consecutive dermatitis patients, in six European dermatological clinics (7). Of the patients tested, 1.2% reacted to oxidized linalool, which means that oxidized linalool is a common allergen in Europe. It also shows that people come in contact with the sensitizing substances in oxidized linalool and that autoxidation is a process that is important to consider. Our research has for many years focused on the effect of autoxidation on the skin sensitizing capacity of various chemicals, among others, terpenes. This is yet another example of

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the importance of examining the fate of a substance in contact with air.

Acknowledgment. The skillful technical assistance of Kerstin Magnusson and Petri Karhunen is gratefully acknowledged, and we thank Prof. Peter Ba¨ckstro¨m for fruitful discussions on photochemistry. This work was financially supported by the Quality of Life and Management of Living Resources program of the European Commission under the key action Environment and Health - Contract QLK4-CT-1999-01558 “Fragrance chemical allergy: a major environmental and consumer health problem in Europe”.

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